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Hamish Collin, Rick Cosstick, Meriel Jones, Brian Tomsett
Angela Tregova and Jill Hughes
Acknowledgement: Mark Wilkinson, protein purification facilities
Molecular Analysis of Flavour biosynthesis in garlic
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Biosynthetic Pathway SO42-
SO32-
S2-cysteine
glutathione
S-methyl-γ-glu-cys
gly
S-methylcysteine
methiin
glutrans-peptidase
oxidase
S-2-CP-γ-glu-cys
gly
S-trans-1-propenyl-γ-glu-cys
S-trans-1-propenylcysteineoxidase
trans-peptidaseglu
HCOOH
S-trans-1-propenylcysteine sulphoxide(isoalliin)
S-methylglutathioneS-(2-carboxypropyl)-glutathione
valine & methacrylate
S-allylglutathione
S-allyl-γ-glu-cys
gly
glutrans-peptidase
Allyl group(source ?)
serine
oxidase
S-allylcysteine
S-allyl-cysteine sulphoxide(alliin)
serine
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What we have done……
• Investigation of intermediates in the pathway
• Identification of key compounds
• Purification of a key enzyme• Allylcysteine synthase
• The search for genes involved in flavour biosynthesis:
2 chloroplastic cysteine synthases1 cytosolic cysteine synthase1 S-allyl cysteine synthase+ 1 cytosolic serine acetyl transferase
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Key observation
Callus convertsallyl thiol to allyl cysteine & alliin
CH2CHCH2-SH (+ O-acetyl-serine ?)
CH2CHCH2-S-CH2CHNH2COOH
CH2CHCH2-S-CH2CHNH2COOH
But not allyl alcoholCH2CHCH2-OH
=
O
XXBut this is not species-specific
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Allyl Cysteine Synthase?
Is there a specific cysteine synthase homologue?
Cysteine synthases do a range of reactions
in other organisms
Sulphide + O-Acetyl Serine Cysteine
Allyl thiol + O-Acetyl Serine Allyl Cysteine
Cysteine synthase
Allyl Cysteine synthase
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Cysteine synthase activity. Q-Sepharose. 7.11.01
00.05
0.10.15
0.20.25
0.3
Fraction
OD
cysteine synthaseactivity
Many fractions show cysteine synthase activity
Protein purification: Ion Exchange chromatography
Garlic leaves were fractionated with ammonium sulphate then separated by ion-exchange chromatography.
Only a few fractions show allyl cysteine synthase activity
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Protein purification: Hydrophobic Interaction Chromatography
Allyl cysteine synthase and cysteine synthase activity co-elute
Phenyl sepharose fractionation
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
1 3 5 7 9
11
13
15
17
19
21
23
25
27
29
31
33
35
37
39
Fraction
OD
55
0
cysteinesyntase activity
allyl cysteinesynthaseactivity
Cysteine production was assayed colorimetrically and allyl cysteine by HPLC
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Protein purification
SDS-PAGE shows a distinct band in the allyl cysteine synthase active fractions at approx. 34 kd
Molecular weight consistent with plant cysteine synthase monomers found previously Fractions 26 27 28 29 30
34000
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What is the Enzyme?
Extract 34000 band and digest with trypsin
- the resultant peptides separated by preparative HPLC
Three selected peptides were sequenced:-
…….FLGVMPSHYSIE………. YLGADLALTDTN………… ……………………SANPGAHYATTGP………….
A simple BLAST search of these peptides in the protein database shows most similarity to a cysteine synthase from Oryza sativa (Rice)
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Probe for S-allyl-CSase
A B C D E F G H I
Peptide 1 2 3cDNA fragments PCR amplified with degenerate primer A – I from the cDNA library
Peptide sequences:
1. FLGVMPSHYSI
2. YLGADLALTDT
3. ANPGAHYA
…. to find the gene and related genes
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AllylCSase aligns with rice sequences
Partial protein sequences relative to Arabidopsis (C) sequence
RCS2 IGLVLVAVQ-KGYRFIAVMPAKYSLDKQMLLRFLGAELILTDPA-IGFNG—MMDKVEELRCS4 IGVAYNALL-KGYRFVAVMPAEYSLDKQMLLTYLGAEVILTDPT-LGFQGQ-LDKVEQIGCS4 IALAYI-GLKKGYKFLGVMPSHYSIERRMLLKYLGADLALTD-TNLGFKG-VLDKVAEL
I KGY F VMP YS MLL LGA LTD GF G DKV
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Proposed Serine Pathway
Important enzymes:
1 SAT/CS complex
2 Free CSase
3 S-allyl-CSase + ?
4 OxidaseL-Serine OASAllyl-source
S-allyl-L-Cysteine
Sulfide
Alliin
Cysteine
1
2
3
4
Acetyl CoA
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cDNA library screening
gsat1 - cytosolic SATase
gcs1 - putative plastidic CSase
(pseudogene)
gcs2 - putative plastidic CSase
gcs3 - cytosolic CSase
gcs4 - putative S-allyl-CSase
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What next ?
• Where are the genes expressed in garlic?• Northerns
• Does the gene encode allylcysteine synthase?• How do we prove it?
• What does it do in planta?• Transformation
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Northern blot analysis1 2 3 4 5
gcs4
gcs3
gcs2
gsat1
18s
1. 7 degree C stored clove
2. RT stored clove3. Sprouting clove4. Leaf5. Root
S-allyl CSase and
the SATase
are expressed in most tissues examined.
The cytosolic CSase is root specific.
Expression for the putative plastidic CSase is uniformly low.
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Is this allylcysteine synthase?
Proof requires expression of the gene and phenotypic testing
• Garlic? This would be best but…..time ?
• E. coli? Does it function alone? In vitro testing only?
• Heterologous plant system? Time ? Arabidopsis ?
• Plant tissue culture? Quick and could form complexes allowing tests in planta
Ideal Choice ?
A quick assessment could mean that we can plan the alternative
If we use ethanol-regulated expression, then we can test the effect on the cellular phenotype of the expression of the
allylcysteine synthase vs. its absence !
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Why ethanol-regulated expression?
alcR transgene tpCAMV35S palcAt
AlcR
AlcR EcDNA
E
+ Ethanol
alc is a simple two component system
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Does it work?
Real time Luciferase Imagingin Arabidopsis
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wt
AGS
LUC 1-12
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1 hour before induction
wt
AGS
LUC 1-12
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Time of induction
wt
AGS
LUC 1-12
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30 minutes after induction
wt
AGS
LUC 1-12
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1 hour after induction
wt
AGS
LUC 1-12
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1.5 hour after induction
wt
AGS
LUC 1-12
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2 hours after induction
wt
AGS
LUC 1-12
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2.5 hours after induction
wt
AGS
LUC 1-12
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3 hours after induction
wt
AGS
LUC 1-12
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3.5 hours after induction
wt
AGS
LUC 1-12
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4 hours after induction
wt
AGS
LUC 1-12
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4.5 hours after induction
wt
AGS
LUC 1-12
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5 hours after induction
wt
AGS
LUC 1-12
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6 hours after induction
wt
AGS
LUC 1-12
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7 hours after induction
wt
AGS
LUC 1-12
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7.5 hours after induction
wt
AGS
LUC 1-12
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8 hours after induction
wt
AGS
LUC 1-12
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11 hours after induction
wt
AGS
LUC 1-12
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13 hours after induction
wt
AGS
LUC 1-12
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Real time ArabidopsisLuciferase Imaging
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Time of induction
wt
AGS
LUC 1-12
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8 hours after induction
wt
AGS
LUC 1-12
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Functional analysis of plant cell cycle genes
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At progeny of AmcycA20 x alcRalcAGUS
A B C D E F
G H I J K L
A B C D E F
G H I J K L
Induced
Non-induced
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 A B C D E F G H I J K L
1.6 kb 1 kb
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 A B C D E F G H I J K L
1 kb
500 bp
A B C D E F G H I J K L 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1 kb
500 bp
AmcycA20 PCR
GUS PCR
alcR PCR
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RT-PCR of AmcycA20 & controls
1 2 3 4 5 6 7 8 9 10 11 12 13 14
A B C D E A B C D E
1 kb
plus RT
1 kb
1 2 3 4 5 6 7 8
A B C D E
Induced RNA minus RT
Total RNA extractedfrom plants ofA = cyclin A20B = HA-tagged cyclin A20 C = siblingD = wild typeE = AGS-1-3
Total DNA extracted fromInduced plants ofA = cyclin A20B = HA-tagged cyclin A20 C = sibling (cyc+;GUS-)D = wild typeE = AGS-1-3 13 = A.majus genomic DNA
There is no DNA contamination
1 2 3 4 5 6 7 8 9 10 11 12 13 14
A B C D E A B C D E
1 kb
Induced plants
Uninduced plants
cycA20 message is specific to
induced plants containing both
T-DNAs
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Western Blots of HA tagged cycA20 WT I N WT I N
Probe = antibody to HA tag
48 kDaHA-CycA20
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Phenotypic analysis Rosette leaves
1 2
Leaf cell density, primary leaf area, rosette leaf number, trichomes and flowering time.
Plants were grown for six weeks.
Vertically grown A.thaliana plants, growing in a tissue culture square plate.
Root growth experiments (after 15 days) and fresh weight measurements (after four weeks).
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Fresh Weight
0
20
40
60
80
100
WT AGS SIBLING CYCA20-HA CYCA20
NON-INDUCED INDUCED
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Root Length – AmcycA20 expressionWT 25 26 27 28 29 30 31 32 AGS
G A A
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Root length
0
1
2
3
4
5
6
7
8
9
0 5 10 15
Days growth
Ro
ot
len
gth
(c
m)
WT
AGS
SIBLING
CYCA20-HA
CYCA20
WT
AGS
SIBLING
CYCA20-HA
CYCA20
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Leaf number and area
sibling
Cyclin A20
Leaf number remains constantafter AMcycA20 expression
Leaf area is bigger after induction in
AmcycA20 expressing lines
Minus ethanol
Minus ethanol
Plus ethanol
Plus ethanol
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Leaf Area
0.00
0.50
1.00
1.50
2.00
2.50
WT AGS Sibling CycA20-HA CycA20
Non-induced Induced
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Cell Size and density
HA-tagged cyclin A20
Uninduced Induced
There appear to be less cells per unit area - cells are larger
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Trichomes on rosette leaves
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Flowering time of cycA20 and controls
05
10152025
WT AGS Sibling CycA20 CycA20
Non-induced Induced
Day
s
Mean flowering time (days) of twenty seedlings of each of HA-tagged cyclin A20, cyclin A20, wild type (Columbia), AGS-1-3 and sibling plants in comparison between non-induced and induced conditions. The plants were induced after 5 days of germination, when the plants reached the 2 leaf-stage, and were checked regularly until the appearance of the first visible flower bud.
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Tobacco transformation for protein expression
RB t35S palcAGarlic gene LB pAg7nptIIpnos
Transformed Untransformed Transformed
kanR
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Tobacco transformation for protein expression
• The tobacco cells can be multiplied in liquid culture
• Induce protein expression
• Determine whether –SH content of cells has increased
• Assay for allylCSase activity
• Use HPLC to look for allylcysteine and ….?
Does tobacco possess an oxidase to make alliin?
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Diagnostic PCRs for transgenic BY2 lines
1 2 3 4 5 6 7
PCR primers:
1.palcA forward
2. t35S reverse
Lane 1 = untransformed BY2
Lane 2 = gcs3 plasmid control
Lane 3 = gcs3 transformant
Lane 4 = gcs4 plasmid control
Lane 5 = gcs4 transformant
Lane 6 = gsat1 plasmid control
Lane 7 = gsat1 transformant
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However…….
Cysteine synthase assays
No detectable increase in cysteine. Time course assays and assay optimisations failed.
S-allyl-cysteine synthase assay (HPLC)
No detectable levels of S-allyl-cysteine.
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Northern blot analysis
gcs3
gcs4
gsat1
Garlic RNA Tobacco RNA
RNA extracted from transgenic tobacco cells after 1, 3 and 6 days induction.
Northern blots show no transgene expression, except gcs3 that was detected after several days induction.
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Why are the transgenes not expressed?
Are there mistakes in the binary constructs?Re-sequencing verified correct assemblies.
Is the alcR cDNA present in the tobacco cell-line? alcR confirmed by PCR.
Is alcR expressed?
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Is alcR expressed?
1 2 3 4 Lane 1 = alcR control (genomic DNA)
Lane 2 = gcs3 BY-2 transformant
Lane 3 = gcs4 BY-2 transformant
Lane 4 = gsat1 BY-2 transformant
RT-PCR results:
No alcR expression detected in any of the transformed cell lines!
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Repeat BY-2 transformation
New BY-2 cell-lines from the John Innes Centre
Transformations have been repeated and we
are currently waiting for new transformants to grow
But is alcR expressed in the new cell-line?
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alcR expression in the new cell-line
Positive RT-PCR controls using degenerate primers that anneal to SAT.
1 2 3 RT-PCR results:Lane 1 = No RT control
Lane 2 = alcR control (genomic DNA)
Lane 3 = alcR expression in the new cells
Again, no detectable alcR expression in the new cell line!
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RT-PCRs using a highly sensitive detection
1 2 3 4 5 6 7 8 9
RT-PCR results:
Lane 1 = untransformed BY-2
Lane 2 = gcs3 BY-2 transformant
Lane 3 = gcs4 BY-2 transformant
Lane 4 = gsat1 BY-2 transformant
Lane 5-8 = No RT controls
Lane 9 = alcR control (genomic DNA)
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RT-PCRs using a highly sensitive detection
1 2 3 4 5 6 7 8 9
RT-PCR results:
Lane 1 = untransformed BY-2
Lane 2 = gcs3 BY-2 transformant
Lane 3 = gcs4 BY-2 transformant
Lane 4 = gsat1 BY-2 transformant
Lane 5-8 = No RT controls
Lane 9 = alcR control (genomic DNA)
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RT-PCRs using a highly sensitive detection
1 2 3 4 5 6 7 8 9
RT-PCR results:
Lane 1 = untransformed BY-2
Lane 2 = gcs3 BY-2 transformant
Lane 3 = gcs4 BY-2 transformant
Lane 4 = gsat1 BY-2 transformant
Lane 5-8 = No RT controls
Lane 9 = alcR control (genomic DNA)
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Future ?
The longer route looks more attractive !• E. coli – his-tagged protein
purification
assay in vitro
• Arabidopsis - test for expression
assay in vivo
phenotype
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Expression of a wheat CSase in tobacco
A. Transgenic tobacco shows 2-fold higher Cys content.
B. SO2 fumigation increased thiol levels.
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Deliverables
• Genes for CSO synthesis enzymes (36m)
• Publication on regulation of S biochemistry in garlic (36m)
• Paper on characterising enzymes in alliin biosynthesis, and alliinase expression, and regulation of sulphur biochemistry in garlic (48m)
• Paper on S pathway genes on production of flavour precursors in garlic (48m)
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Thanks to ……..
LiverpoolAngela TregovaJill Hughes
Piyarat Parinyapong
Hairul Roslan
Chris WoodMike White
Mark CaddickBrian Tomsett
Jealott’s HillJackie PaineMary KnightSusan WrightJustin SweetmanAlberto MartinezWolfgang SchuchAndy GreenlandIan Jepson
ICI AgrochemicalsICI SeedsZeneca SeedsZeneca AgrochemicalsSyngenta
JICJohn Doonan and his lab
FundingBBSRCEU FP5 Garlic &
Health